The Na,K-ATPase is an important membrane-associated enzyme responsible for maintaining the high internal potassium concentration and low internal sodium concentration characteristic of most animal cells. The ion gradients created by the Na,K-ATPase are fundamental to such diverse cellular functions as the regulation of cell volume and pH, the uptake of nutrients and membrane excitability. The Na,K-ATPase belongs to a widely distributed class of P-type cation transporting proteins that includes the intracellular and plasma membrane Ca-ATPases, and the H,K-ATPase of gastric mucosa. All these P-type ATPases have similar structures and common reaction mechanisms. However, in addition to having different cation affinities these P-type ATPases can have different intracellular locations. Depending on the specific ATPase, these enzymes can be found at the plasma membrane or in cytoplasmic vesicles or organelles. The purpose of this project is to gain insight into the role of the individual Na,K- ATPase subunits in directing the assembly, cellular sorting and function of the enzyme. Using baculovirus-infected insect cells we have shown that the alpha-subunit has activity independent of the beta-subunit. This activity will be further characterized to determine if the unassociated alpha-subunit can mediate the occlusion and transport of ions. In addition, the properties of the naturally occurring truncated alpha isoform, alpha1T will be determined. To identify and characterize the regions responsible for the assembly and intracellular transport of the Na,K-ATPase, chimera alpha subunits consisting of the Na,K-, H,K- and Ca- ATPases will be constructed. These chimeric subunits will be used to characterize the intracellular transport of the P-type ATPases in insect and mammalian cells. To ascertain the domains important for the transport of ions and binding of inhibitors, the chimeric proteins will be characterized with respect to ion and inhibitor specificity. These studies should help in defining the role of the individual subunits and of specific domains of the subunits in the assembly, intracellular transport and function of the Na,K-ATPase.